The purpose of our project is to study the relationship of erythrocyte metabolism with the plastic properties of the membrane. This information would then contribute to the understanding of the pathophysiology of hemolytic diseases, the red blood cell storage lesion, and the biological properties of plasma membranes in general. Our plan is to focus in part on the phenomenon of endocytosis in white ghosts. It appears that induction of spectrin-free areas of the membrane is a prior requirement for endocytosis or exocytosis. We propose to specifically test this hypothesis by using a ferritin-labeled anti-spectrin antibody which should allow us to detect movement of spectrin in the plane of the membrane during the endocytosis process. In order to further understand membrane structure and determine if there are microenvironments in the membrane, erythrocyte membranes prepared by hypotonic lysis and by the glycol isotonic method will be sheared, and the resulting microvesicule classes will be harvested and assessed for their membrane polypeptide content as well as the enzyme activities of acetylcholinesterase (a marker of the membrane exterior), NASH oxidoreductase and glyceraldehyde phosphate dehydrogenase (markers of the cell interior) and the three major ATPases which are indicators of the persistence of overall cell membrane integrity. In pursuing our prior studies on the movements of 45Ca within intact erythrocytes, we are attempting to identify those peptides which 45Ca appears to be bound when it moves membrane associated pools. Studies of the erythrocyte membrane storage lesion will be pursued in collaboration with our colleagues at the Lederman Army Institute for Research, where we will be attempting to correlate in vivo survival of erythrocytes stared under a variety of conditions with endocytosis induced by primaquine and chlorpromazine in intact erythrocytes.